This invention relates to a method of purifying biological material produced by cells. More particularly, the invention relates to a process for recovering nonsecreted substances produced by cells free of many unwanted high molecular weight contaminants.
Advances in cellular biology have demonstrated that the concentration of a number of active biological substances is substantially greater inside the cell than in the surrounding media. These substances, whether because of their molecular weight, charge, or other reasons, are not easily transported across the cell membrane or are simply not secreted by the cell. Any process for recovering these products requires disrupting or lysing the cell membrane. Upon cell lysis, the media is contaminated with cell fragments and unwanted high molecular weight contaminants. Since the concentration of the non-secreted substance is relatively small compared with the total concentration of cell components, recovery of the substance of interest may be difficult. The phrase nonsecreted substance, as used herein, means a substance which is produced in significant recoverable quantities within the cell which is not secreted or only partially secreted.
An associated problem is the release from cells of pyrogens; fever-inducing substances, during cell lysis. Many prokaryotic bacteria, particularly the gram negative enterobacteria, produce endotoxins which are pyrogenic. Since these gram negative bacteria, e.g., E. coli, bacillus, and pseudomonas are the basic bacteria used in DNA recombinant technology, the pyrogenic endotoxins produced by these bacteria are a serious problem. These pyrogenic substances are primarily lipopolysaccharides having molecular weights greater than 5.times.10.sup.4 daltons. A number of enzymes and other nonsecreted substances of interest have lower molecular weights than the pyrogens, so a method for molecular sieving of contaminants released upon cell lysis would assist in purification and recovery.
U.S. Pat. No. 4,352,883 discloses a method for producing microcapsules containing viable cells. Copending U.S. application Ser. No. 372,835 now U.S. Pat. No. 4,409,331 takes this encapsulation procedure a step further and discloses a process for producing substances produced by cells. More particularly, the U.S. Pat. No. 4,409,331 discloses that low molecular weight substances secreted by cells can diffuse through the semipermeable membrane of the microcapsule and can be collected in the extracapsular media with a minimum of contamination and that higher molecular weight substances secreted by cells can be trapped within the microcapsules and can be collected by disrupting the capsule membrane without lysing the cells.
Accordingly, an object of the invention is to provide a method for recovering nonsecreted substances substantially free of contaminants. Another object of the invention is to provide a method for substantially pyrogen-free recovery of nonsecreted substances from pyrogen-producing microorganisms. A further object is to provide a method of lysing the cell membranes of encapsulated cells without disrupting the membrane of the surrounding microcapsule. A still further object of the invention is to provide a method for recovery of low molecular weight, nonsecreted substances from genetically modified bacteria.
These and other objects and features of the invention will be apparent from the following description and from the drawings.